Effect of Foliar Sprays of Sulfuric Acid and Thiourea on Herbage and Seed Yield in Berseem (Trifolium Alexandrinum L.)

Berseem (Trifolium alexandrinum L.) is an important legume fodder crop grown for livestock in Asia, Africa, USA and Australia. In India berseem is grown in Rabi (winter) season and it occupies 2 million ha area (Kumar et al., 2013). Berseem is generally grown as pure stand by the farmers for feeding to the livestock. During severe winter months periods from December to February, berseem growth is slow due to low minimum air temperature or cold wave or frost occurrence which affects fodder availability to the livestock. To increase growth of berseem either in pure stand or in mixture, farmers generally use excessive urea which not only affects nodule development in roots of legumes but also pollute the environment (Dogra and Dudeja, 1993). Mixture of berseem clover with cereal crop enhances total dry matter yield, improves fodder quality, reduces fertilizer use and also increases subsequent crop yield (Ross et al., 2004). Farmer harvests 3-4 cuttings of berseem up to late mid April as fodder for livestock and then leaves the berseem crop for seed production. After 3-4 cuts as green fodder, berseem plants retain less foliage, poor flowering and finally low seed production (Kumar et al., 2013). It is very essential that berseem crop attains sufficient height and tillering capacity during initial cuts for obtaining high fodder yield and later on crop left for seed production produces good quantity and quality of seed. Abiotic stress factors cause extensive loss to agriculture production worldwide (Anitha et al., 2006; Kumar et al., 2013; Wahid et al., 2017). Among abiotic stress factors, low temperature or frost causes considerable reduction in growth and development of berseem leading to reduced availability of green fodder. Measures for alleviating cold injury in crops so far are confined to the application of irrigation at large (Rao and Sahu, 1991). But in berseem and its intercrops, frequent irrigation impede root growth and causes diseases in crop due to more damp conditions and also make difficult to harvest fodder. Chemical means of assuage cold or low temperature injury, therefore, appears necessary for optimizing berseem and its intercrops productivity during low temperature condition. Thiourea (TU) is an organic compound having molecular formula Cs(NH₂)₂ has been identified as an effective bio-regulator to improve growth of plants under stress condition (Pandey et al., 2013; Amin et al., 2014; Singh, 2015). Foliar spray of TU improves vegetative growth and dry matter production in legume (Dayanand et al., 2013; Amin et al., 2014), enhances carbohydrate and nitrogen metabolism (Singh 2015), improve photosynthetic efficiency (Pandey et al., 2013), promote synthesis of auxin and cytokinin for the enhancement of cell division and chlorophyll formation under stress conditions (Amin et al., 2014). Sulphur from H₂SO₄ promote oil synthesis and enhances seed protein, amino acids, haematin enzymes (such as catalase and peroxidase) and improve chlorophyll formation (Singh, 2015). In the present study, the selected chemicals have different characteristics, easily available and economical which make them suitable choice for mitigating the damage induced by low temperature stress. So, keeping this objective in view, a field experiment was designed to know the effect of different bio-regulators sprays during early cuts on the growth of berseem grown as pure stand and in mixture against very low minimum air temperature/frost injury and seed production of berseem under different sowing conditions.

Plant height and tillers plant^-1
 
Plant height at different cuts of berseem pure and in mixture with oat is presented in Table 1. Data pertaining to berseem plant height and tillers plant^-1 under different treatments was recorded at 2^nd, 3^rd, 4^th and at harvest while oat plant height and tillers were recorded at 2^nd and 3^rd cuts only owing its cessation after that. No significant difference in plant height of berseem was recorded in berseem pure and berseem+oat mixture during the 2^nd, 4^th cuts and at harvest. During 3^rd cut, berseem plants in pure stand gained significantly more height than in mixture (Table 1) owing to no competition from other crop. Similarly significantly more number of tillers plant^-1 in berseem were recorded in pure stand than in mixture during 3^rd, 4^th cuts and at harvest except at 1^st cut. Higher numbers of tillers plant^-1 in pure stand might be due to better availability of growth resources to the plants. As oat was present only in mixture (berseem + oat) data related to its plant height and tillers plant^-1 is not presented in Table 1.
 

       
Foliar sprays of different bio-regulators recorded significant effect on plant height and tillers plant^-1 of berseem and oat (Table 2). Foliar sprays of thiourea @ 0.05% recorded taller plants and more tillers plant^-1 in berseem and oat at different cuts compared to corresponding untreated control. Foliar application of 0.1% H₂SO₄ also recoded significant increase in plant height and tillers plant^-1 of berseem and oat over control. The increase in growth parameters of berseem and oat with exogenous application of thiourea might possibly be due to increased translocation of sucrose metabolites from source to sink, improved plant growth potential due to enhanced photosynthetic efficiency, enhancement of cell division and chlorophyll formation (Amin et al., 2014; Singh, 2015; Anitha et al., 2006). Many researchers had also reported positive effect of exogenous application of thiourea in chickpea (Cicer arietinum), faba bean (Vicia faba), wheat (Triticum aestivum L.) and maize (Zea mays L.) crops (Dayanand et al., 2013; Amin et al., 2014; Pandey et al., 2013; Singh, 2015). Exogenously applied thiourea also imparts stress tolerance and has been reported to increase the growth and development of plants when the conditions are not favorable (Pandey et al., 2013) hence a favorable effect of its application on growth and development of berseem and oat had been observed in our study during low temperature condition. Sharma and Jain (2003) and Jat et al., (2014) also recorded positive effect of foliar application of sulfuric acid (0.1%) on growth attributes of Indian mustard (Brassica juncea) and chick pea under low temperature injury in arid irrigated conditions.
 

 
Green fodder yield (GFY) and dry matter yield (DMY)
 
Production of greater forage and dry matter yield per hectare is very important for the producers. Data manifested in Table 3 shows that intercrop mixtures and application of different chemicals as foliar sprays had significant effect on the green fodder yield (GFY) and dry matter yield (DMY) during various cuts. Noticeable increase in green fodder and dry matter yield was obtained in 1^st, 2^nd and 3^rd cuts in berseem + oat intercrop over berseem pure stand where as in the 4^th cut both these parameters were at par with each other. Highest green fodder and dry matter in 3^rd cut was due to favorable temperature for growth of the crops where as lower temperature affected its growth in previous cuts. In respect to total green fodder and dry matter yield, berseem + oat mixture recorded significantly higher respective values by 7.0% and 6.1 % than pure stand of berseem (Table 3). The benefits of intercropping berseem clover with cereal crops, include increased total green fodder and dry matter yield, improved forage quality (Ross et al., 2004), reduced fertilizer requirements and increased subsequent crop yield (Dogra and Dudeja, 1993). Mixture also leads to more efficient absorption of nutrients from the soil, more interception of light energy at different layers and finally more photosynthetic rate, better translocation of photosynthates from source to sink leading to higher green fodder and dry matter yield.
 

        
Foliar sprays of different bio-regulators on crop significantly affected green fodder and dry matter yield during (Table 3). Among different chemical foliar treatments, foliar sprays of thiourea and H₂SO₄ recorded significantly higher green fodder and dry matter yield than control. Foliar spray of 0.05% thiourea on crop recorded 11.2%, whereas dilute sulfuric acid at 0.05 and 0.1% led to 5.2 and 8.0% increase in green fodder yield over control. The increase in green fodder yield with foliar application of 0.1% TU was less compared to 0.05% TU but was significantly better than control. Similarly, highest total dry matter yield with foliar sprays of 0.05% TU was significantly better than foliar sprays of 0.05, 0.1% H₂SO₄ and control. The increase in total DMY with foliar sprays of 0.05% TU was to the tune of 11.4, 5.5 and 2.8% with respect to control, foliar sprays of 0.05% and 0.1% H₂SO₄, respectively. The increase in GFY and DMY in 2^nd, 3^rd and 4^th cut as well as total with foliar sprays of different bio-regulators may be attributed to enhanced growth of crop in subsequent cuts after 1^st cut due to maintenance of redox state of the cell under stress conditions (Sharma and Jain, 2003; Anitha et al., 2006; Kumar et al., 2013). Singh (2015) and Sharma and Jain (2003) also reported significant improvement in the potato and mustard crop yield with foliar application of 0.1% TU and concentrated H₂SO₄ (1.0 ml/L water) over control due to crop protection from the low temperature. Further, sulfhydryl compound play role in improving dry matter partitioning and thus improve the productivity. Garg et al., (2006) reported that thiourea (TU) as foliar spray on the plant significantly improved plant height, leaf area, dry matter production, net photosynthetic rate and concentration of total chlorophyll and starch in the leaves compared to control. The slight reduction in green fodder and dry matter yield at higher concentration of TU might be due to reduction in soluble protein in plants as higher concentration stresses the plant as reported by Mani et al., (2013).
 
Crude protein (CP) content and crude protein yield (CPY)
 
Crude protein content and crude protein yield are important parameters for determining the quality of the fodder produced. Mixtures had significant effect on the protein content and crude protein yield of the forage produced (Table 4). During 1^st and 2^nd cut pure berseem crop recorded significantly higher protein content than berseem + oat mixture intercrop. However, 3^rd and 4^th cut registered non significant differences in respect to crude protein content which might be due to low or no presence of oat crop in these cuts. Average crude protein content in berseem pure crop was 5.3% higher than berseem + oat mixed crop. With respect to crude protein yield (CPY), berseem + oat mixture in 2^nd and 3^rd cut recorded significantly more crude protein yield possibly due increase in green fodder and dry matter yield (Table 3). Berseem + oat mixture registered significant increase in total crude protein yield by 3.4% over berseem pure stand. Zhang et al., (2015) also advocated that forage intercropping (alfalfa+ rye grass) system exhibited higher DM and CP yields than the sole alfalfa forage crop.
 

       
Foliar sprays of bio-regulators had also significant effect on crude protein content and crude protein yield of green fodder produced during different cuts (Table 4). Significant improvement in crude protein content of fodder was recorded with foliar sprays of 0.05% TU over foliar application of 0.05% H₂SO₄ and control during 2^nd, 3^rd and 4^th cut. Average crude protein content was improved by 6.3% over control with foliar application of 0.05% TU. Similarly crude protein yield (CPY) was also influenced significantly with application of bio-regulator. Highest CPY was obtained with foliar sprays of 0.05% TU during 2^nd, 3^rd and 4^th cut and was found statistically better than 0.05 and 0.1% H₂SO₄ and control. With the 0.05% TU, 0.05 and 0.1% H₂SO₄ application, increment in total CPY was 19.1%, 9.2% and 6.6% over control. Pandey et al., (2013) also recorded 2.5% and 19% increase in protein and oil content of TU treated mustard plant over control due to increased total uptake of N P and K from soil. Higher concentration of TU (0.1%) significantly reduced crude protein content during 2^nd and 3^rd cut while significant reduction in CPY with higher dose of TU was recorded in 3^rd cut only (Table 3). Mani et al., (2013) also reported significant reduction in soluble protein in potato plants treated with 1000 nM TU over 250 nM suggesting higher concentration of TU stresses the plant due to low transfer of photosyntheates in sink organ. However, in our study average crude protein content and total CPY were found to be statistically similar with foliar sprays of 0.05% and 0.1% TU.
 
Yield attributes and berseem seed yield: In our study, fodder mixture and bio-regulators influenced berseem yield attributes, biological yield and seed yield significantly (Table 5). Considerably higher numbers of pod plant m^-2, 1000-grain weight, biological yield, seed yield and harvest index were recorded in berseem pure stand than berseem+ oat intercrop mixture. Increase in pods m-2 was by 4.4% while 1000-grain weight was improved by 7.2% in berseem pure crop over mixture. Pure stand of berseem recorded 7.9% higher biological yield than mixed berseem and oat. Seed yield is important criterion for realizing better return in any crop. In our study, seed yield of berseem was significantly improved in berseem pure stand than berseem+ oat mixture crop (Table 5) possibly due to better plant stand. The results were in line with Shoaib et al., (2013) who reported that higher yield production of Egyptian clover at each growth stage was due to sufficient berseem plant size at first cut that ensured more reserves stored in lower part of the plant that helped high re-growth as compared to Egyptian clover in mixture. Harvest index was significantly superior in berseem pure stand than berseem + oat mixture (Table 5) possibly due to higher biological and seed yield because of better growth of plants.
 

       
Different foliar treatments of thiourea and dilute sulfuric acid recorded significantly more berseem pods m^-2, seeds pod^-1, 1000-grain weight, biological yield and seed yield over control (Table 5). Application of H₂SO₄ and TU increased the mean head density by 7.7-14.4% over the control treatment. The highest heads m^-2 were recorded with foliar application of 0.05% TU which was statistically not different from H₂SO₄ and TU (0.1%) but significantly better than H₂SO₄ 0.05% and control. Jat et al., (2014) also reported significant improvement in pods plant^-1 with foliar sprays of H₂SO₄ on chickpea during low temperature conditions. All the foliage applied bio-regulators recorded significantly higher numbers of seeds head^-1 (except 0.05% H₂SO₄) than control. Foliar application of 0.1% TU and 0.1% H₂SO₄ produced 19.0% and 11.9% more seeds head^-1 than control. Latha and Nadanassababady (2003) also reviewed that two foliar sprays of 0.1% sulfuric acid and Thiourea at 45 days after sowing and 15 days after first spray significantly improved the yield attributes of mustard. Thousand-seed weight was significantly higher with the foliar sprays of the TU 0.05% compared to the control. Thiourea application caused 4.8%-7.8% increase in thousand-grain weight of the berseem (Table 5). Sulfuric acid foliar application caused little increase in 1000-grain weight, but differences were insignificant. The increase in translocation of photosyntheates to the plant and further to seed under foliar sprays of Thiourea as reported by Pandey et al., (2013) might be responsible for increasing the 1000-seed weight in our study. Increase in biological yield (seed + straw) was recorded with foliar sprays of different bio-regulators (Table 5). Maximum biological yield of 64.0 q ha-1 was recorded in 0.05% TU treated plants followed by 0.1% TU treated plants. The increase in biological yield with 0.05% was to the tune of 28% over control possibly due to better growth and development of the crop as apparent by increased plant height and tillers plant^-1 (Table 1). Further, foliar sprays of sulfuric acid (0.05 and 0.1%) caused significant improvement in biological yield over control. As earlier discussed that sulfhydryl compound from H₂SO₄ and TU helps in improving plant height, leaf area, net photosynthetic rates, concentration of total chlorophyll and starch in the leaves enhancing dry matter partitioning and thus improving the productivity (Garg et al., 2006; Dayanand et al., 2013; Kumar et al., 2013).
       
Yield is expression of various morphological, physiological and growth parameters in crop. Foliar sprays of dilute sulfuric acid and TU at different concentrations led to improvement in the seed yield of berseem over control. Maximum increase in berseem seed yield was observed under the foliar sprays of 0.05% TU which was 21.4% more than control followed by foliar sprays of 0.1% TU (17.1%). Foliar sprays of H₂SO₄ (0.05 and 0.1%) helped to increase the seed yield ranging from 9.6-16.9% over control. Singh (2015) also recorded that foliar application of concentrated H₂SO₄ @ 1.0 ml/L of water and 0.1% TU not only protected the potato crop from frost injury but also led to 11.1 and 15.8% increase in the tuber yield. Increase in seed yield of berseem in our study might be due to enhanced growth and tillering of the crops after the foliar application these bio-regulators (Table 1). Higher growth and development in terms of height and tillering under TU treatment at 2^nd cut might had resulted in more food reserves leading to better growth during subsequent cuts and finally in seed yield of berseem. TU can save cells from harm and maintain their structure during stress condition which in turn can lead to more absorption and translocation of minerals from the soil to the plants (Anitha et al., 2006; Pandey et al., 2013; Kumar et al., 2013). Accumulation of more nutrients and reserves in the plant might be responsible for good and healthy plants in later cuts resulting more seeds yield. Higher concentration TU (0.1%) recorded slight decrease in seed yield of berseem than lower concentration (0.05%) possibly due to suppressing of water and mineral absorption by the roots (Mani et al., 2013). Foliar application of different bio-regulators did not have any significant effect on the harvest index.
 
Year’s effect
 
Berseem plant height and tillers differed significantly during the year 2009-10 and 2010-11 (Table 6). Significantly taller and with more tillering capacity berseem plants were recorded during first year study than second. Yadav (2010) also reviewed that cold conditions generally results in stunted growth and reduced tillering in the crops. Similarly, more green fodder yield and dry matter yield was recorded during the year 2009-10 over 2010-11 possibly due to suitable climatic conditions during the first year. As frost occurred for 26 days during the 2010-11, might impeded the growth of plant as evidenced by height and tillers of berseem at harvest resulted in lower generation of photosyntheates  causing low green fodder and dry matter yield. Average crude protein content and total crude protein yield was also significantly low during second year of study. Yadav (2010) also advocated that exposure of the plant to the cold stress causes reduction and impairing of photosynthesis, reduction in protein synthesis and general metabolic process. Significantly higher numbers of pods m^-2 were recorded in 2009-10 probably due to more growth and tillering of plants due to normal weather conditions. Seeds pod^-1 and 1000 grain weight was statistically similar during the both years of study. Biological yield and seed yield recorded 3.9% and 6.7% increase during first year of study than the year 2010-11 possibly due to lesser occurrence of frost days during that year. Kumar et al., (2013) also reported that temperature and rainfall can influence deposition of photosyntheates in the seeds resulting variation in berseem seed production. Harvest index was significantly more during first year of study due to production of more biomass by the crop.
 

The results obtained in the present study showed that mixture of berseem and oat resulted in higher green fodder yield and dry matter yield than berseem pure stand. During harsh winter months when very low minimum air temperature or frost injury occurs, two foliar sprays of thiourea at 0.05% at weekly interval 10 days after 1^st and 2^nd cut is promising in enhancing green fodder and seed yield of berseem grown in pure stand and in mixture with oat. Foliar application of TU resulted in 11.2% and 21.4% increase in green fodder yield and seed yield of berseem. The use of TU as bio-regulatory compounds thus opens up a new avenue for increasing green fodder and seed yield improving the fodder availability for the livestock around the world.